The reproductive system of model species such as the domestic cat and its non-domestic relatives offers an unusual opportunity for understanding the changes and adaptations of genes that mediate species isolation and survival. The Laboratory of Genomic Diversity (LGD) has undertaken a comparative physiology approach to describe the aspects of feline reproduction that discriminate between species and allow for behavioral co-adaptation. In addition, empirical methods to develop cryopreservation have been assessed to optimize assisted reproductive technologies in these species, including artificial insemination, in vitro fertilization (IVF) and embryo transfer between these closely related species.Models of how to use new technologies to assess reproductive fitness are emerging to help insure gene diversity and propagate endangered species. Non-invasive hormone metabolite monitoring assays, artificial insemination techniques and genome resource banking have been developed to aid in studies examining the adaptive differences among the Felidae. Significant discoveries include the finding that standard cooling techniques for cat sperm result in extensive cell membrane damage, allowing the creation of slower, more effective cooling procedures. Sperm from males producing many malformed cells are less likely to survive cooling-freezing-thawing stress. Egg freezing studies reveal that cat eggs are highly sensitive to cool temperatures. Investigations also continue on the transmission of Feline Immunodeficiency Virus (FIV) (related to HIV) in cat semen. A related challenge is the high incidence of abnormally shaped sperm found in the semen of some domestic cats and many endangered cat species. This condition, known as teratospermia and common in men, limits fertilization capacity. Although sperm from teratospermic cats were found to have the same amount of DNA as normal sperm, the former have decreased amounts of protamine, a class of nuclear proteins that play an important role in DNA stabilization. Semen has also been collected and cryopreserved from several wildtype and disease cat models for testing through IVF studiesCats have the short generation times and large litter sizes required for development of transgenic and knock-out research models. Successful adaptation of these techniques to the cat would greatly increase the ability of researchers to develop feline models of human genetic diseases. In collaboration with James Kehler (University of Pennsylvania, School of Veterinary Medicine), we have developed a new method to capture the genetic diversity of existing feline models of human genetic diseases. While working in collaboration with Dr. Ronald McKay at the NINDS, we have succeeded in using retroviral vectors to transduce feline fibroblasts to over-express 4 exogenous human genes sufficient to reprogram them into putative pluripotential stem cell (iPS) lines. These gain of function experiments demonstrate a conservation of function between the human and feline orthologs encoding the transcription factor Oct4 that is required for the maintenance of pluripotency. In addition, Oct4 has also been implicated as an oncogene required to maintain the pluripotentiality of some human testicular germ cell tumors (TGCTs) and is a potential therapeutic target. We are using this strategy of reprogramming to establish iPS lines from several of the unique cat models of genetic diseases being characterized as part of other projects at the LGD. Tissues differentiated from these iPS lines will be useful resources for studying these diseases in vitro. New gene therapies to correct these genetic defects in trans will be tested on these cells first in vitro. In addition, wildtype feline iPS cell lines will be used for gene targeting through homologous recombination and subsequent nuclear transfer or chimeric blastocyst injection experiments to develop new knock-out disease models of cats.